Journal of Plant Growth Regulation

, Volume 24, Issue 3, pp 188–200 | Cite as

The Involvement of Cytokinin Oxidase/Dehydrogenase and Zeatin Reductase in Regulation of Cytokinin Levels in Pea (Pisum sativum L.) Leaves

  • Alena Gaudinová
  • Petre I. Dobrev
  • Blanka Šolcová
  • Ondřej Novák
  • Miroslav Strnad
  • David Friedecký
  • Václav Motyka
Original Article


Cytokinin metabolism in plants is very complex. More than 20 cytokinins bearing isoprenoid and aromatic side chains were identified by high performance liquid chromatography-mass spectrometry (HPLC-MS) in pea (Pisum sativum L. cv. Gotik) leaves, indicating diverse metabolic conversions of primary products of cytokinin biosynthesis. To determine the potential involvement of two enzymes metabolizing cytokinins, cytokinin oxidase/dehydrogenase (CKX, EC and zeatin reductase (ZRED, EC, in the control of endogenous cytokinin levels, their in vitro activities were investigated in relation to the uptake and metabolism of [2−3H]trans-zeatin ([2−3H]Z) in shoot explants of pea. Trans-zeatin 9-riboside, trans-zeatin 9-riboside-5′-monophosphate and cytokinin degradation products adenine and adenosine were detected as predominant [2−3H]Z metabolites during 2, 5, 8, and 24 h incubation. Increasing formation of adenine and adenosine indicated extensive degradation of [2−3H]Z by CKX. High CKX activity was confirmed in protein preparations from pea leaves, stems, and roots by in vitro assays. Inhibition of CKX by dithiothreitol (15 mM) in the enzyme assays revealed relatively high activity of ZRED catalyzing conversion of Z to dihydrozeatin (DHZ) and evidently competing for the same substrate cytokinin (Z) in protein preparations from pea leaves, but not from pea roots and stems. The conversion of Z to DHZ by pea leaf enzyme was NADPH dependent and was significantly inhibited or completely suppressed in vitro by diethyldithiocarbamic acid (DIECA; 10 mM). Relations of CKX and ZRED in the control of cytokinin levels in pea leaves with respect to their potential role in establishment and maintenance of cytokinin homeostasis in plants are discussed.


Aromatic cytokinin cis-zeatin Cytokinin Cytokinin oxidase/dehydrogenase Dihydrozeatin Metabolism Pea trans-zeatin Zeatin reductase 



This work was supported by the Grant Agency of the Czech Republic (206/03/0313). The authors thank Dr. Miroslav Kamínek for critical reading of manuscript and Vanda Lacmanová and Marie Korecká for excellent technical assistance.


  1. Armstrong DJ. 1994. “Cytokinin oxidase and the regulation of cytokinin degradation” In: Mok DWS, Mok MC (eds.). Cytokinins: Chemistry, Activity, and Function. Boca Raton, FL, USA, CRC Press, pp 139–154Google Scholar
  2. Bilyeu KD, Cole JL, Laskey JG, Riekhof WR, Esparza TJ, and others. 2001. Molecular and biochemical characterization of a cytokinin oxidase from maize. Plant Physiol 125:378–386CrossRefPubMedGoogle Scholar
  3. Bilyeu KD, Laskey JG, Morris RO. 2003. Dynamics of expression and distribution of cytokinin oxidase/dehydrogenase in developing maize kernels. J Plant Growth Regul 39:195–203CrossRefGoogle Scholar
  4. Boiten H, Azmi A, Dillen W, De Schepper S, Debergh P, and others. 2004. The Rg-1 encoded regeneration capacity of tomato is not related to an altered cytokinin homeostasis. New Phytol 161:761–771CrossRefGoogle Scholar
  5. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  6. Chatfield JM, Armstrong DJ. 1986. Regulation of cytokinin oxidase activity in callus tissues of Phaseolus vulgaris L. cv Great Northern. Plant Physiol 80:493–499PubMedGoogle Scholar
  7. Corbesier L, Prinsen E, Jacqmard A, Lejeune P, Van Onckelen H, and others. 2003. Cytokinin levels in leaves, leaf exudate and shoot apical meristem of Arabidopsis thaliana during floral transition. J Exp Bot 54:2511–2517CrossRefPubMedGoogle Scholar
  8. Davies PJ, Horgan R, Heald JK, McGaw BA. 1986. Endogenous cytokinins in vegetative shoots of peas. J Plant Growth Regul 4:311–323Google Scholar
  9. Dobrev PI, Kamínek M. 2002. Fast and efficient separation of cytokinins from auxin and abscisic acid and their purification using mixed-mode solid-phase extraction. J Chromatogr A 950:21–29PubMedGoogle Scholar
  10. Dobrev P, Motyka V, Gaudinová A, Malbeck J, Trávníčková A, and others. 2002. Transient accumulation of cis- and trans-zeatin type cytokinins and its relation to cytokinin oxidase activity during cell cycle of synchronized tobacco BY-2 cells. Plant Physiol Biochem 40:333–337CrossRefGoogle Scholar
  11. Emery RJN, Leport L, Barton JE, Turner NC, Atkins CA. 1998. Cis-isomers of cytokinins predominate in chickpea seeds throughout their development. Plant Physiol 117:1515–1523CrossRefPubMedGoogle Scholar
  12. Emery RJN, Ma Q, Atkins CA. 2000. The forms and sources of cytokinins in developing white lupin seeds and fruits. Plant Physiol 123:1593–1604CrossRefPubMedGoogle Scholar
  13. Galuszka P, Frébort I, Šebela M, Sauer P, Jacobsen S, and others. 2001. Cytokinin oxidase or dehydrogenase? Mechanism of cytokinin degradation in cereals. Eur J Biochem 268:450–461CrossRefPubMedGoogle Scholar
  14. Galuszka P, Frébortová J, Werner T, Yamada M, Strnad M, and others. 2004. Cytokinin oxidase/dehydrogenase genes in barley and wheat. Cloning and heterologous expression. Eur J Biochem 271:3990–4002CrossRefPubMedGoogle Scholar
  15. Haberer G, Kieber JJ. 2002. Cytokinins. New insights into a classic phytohormone. Plant Physiol 128:354–362CrossRefPubMedGoogle Scholar
  16. Houba-Hérin N, Pethe C, d´Alayer J, Laloue M. 1999. Cytokinin oxidase from Zea mays: purification, cDNA cloning and expression in moss protoplasts. Plant J 17:615–626PubMedGoogle Scholar
  17. Jäger AK, Stirk WA, Van Staden J. 1997. Cytokinin oxidase activity in habituated and non-habituated soybean callus. Plant Growth Regul 22:203–206Google Scholar
  18. Jameson PE. 1994. “Cytokinin metabolism and compartmentation” In: Mok DWS, Mok MC, (eds.), Cytokinins: Chemistry, Activity, and Function, Boca Raton, FL, USA, CRC Press, pp 113–128Google Scholar
  19. Jones RJ, Schreiber BMN. 1997. Role and function of cytokinin oxidase in plants. J Plant Growth Regul 23:123–134Google Scholar
  20. Kamínek M, Armstrong DJ. 1990. Genotypic variation in cytokinin oxidase from Phaseolus callus cultures. Plant Physiol 93:1530–1538PubMedGoogle Scholar
  21. Kamínek M, Březinová A, Gaudinová A, Motyka V, Vaňková R, and others. 2000. Purine cytokinins: a proposal of abbreviations. J Plant Growth Regul 32:253–256Google Scholar
  22. Kamínek M, Motyka V, Vaňková R. 1997. Regulation of cytokinin content in plant cells. Physiol Plant 101:689–700Google Scholar
  23. Kasahara H, Takei K, Ueda N, Hishiyama S, Yamaya T, and others. 2004. Distinct isoprenoid origins of cis- and trans-zeatin biosyntheses in Arabidopsis. J Biol Chem 279:14049–14054PubMedGoogle Scholar
  24. King RA, Van Staden J. 1987. The metabolism of N6-(Δ2-isopentenyl)[3H]adenine by isolated organs of Pisum sativum. J Plant Physiol 131:181–190Google Scholar
  25. King RA, Van Staden J. 1990. The metabolism of N62-isopentenyl)[3H]adenine by different stem sections of Pisum sativum. J Plant Growth Regul 9:237–246CrossRefGoogle Scholar
  26. Letham DS, Palni LMS. 1983. The biosynthesis and metabolism of cytokinins. Annu Rev Plant Physiol 34:163–197CrossRefGoogle Scholar
  27. Martin RC, Mok MC, Habben JE, Mok DWS. 2001. A maize cytokinin gene encoding an O-glucosyltransferase specific to cis-zeatin. Proc Natl Acad Sci USA 98:5922–5926PubMedGoogle Scholar
  28. Martin RC, Mok MC, Shaw G, Mok DWS. 1989. An enzyme mediating the conversion of zeatin to dihydrozeatin in Phaseolus embryos. Plant Physiol 90:1630–1635PubMedGoogle Scholar
  29. Massonneau A, Houba-Hérin N, Pethe C, Madzak C, Falque M, and others. 2004. Maize cytokinin oxidase genes: differential expression and cloning of two new cDNAs. J Exp Bot 55:2549–2557CrossRefPubMedGoogle Scholar
  30. Mok DWS, Mok MC. 2001. Cytokinin metabolism and action. Annu Rev Plant Physiol Plant Mol Biol 52:89–119CrossRefPubMedGoogle Scholar
  31. DWS, Mok MC, Shaw G, Dixon SC, Martin RC. 1990. “Genetic differences in the enzymatic regulation of zeatin metabolism in Phaseolus embryos” In: Pharis RP, Rood SB (eds.), Plant Growth Substances 1988. Berlin, Germany, Springer-Verlag, pp 267–274Google Scholar
  32. Mok MC. 1994. “Cytokinins and plant development—an overview” In: Mok DWS, Mok MC (eds.), Cytokinins: Chemistry, Activity, and Function. Boca Raton, FL, USA, CRC Press, pp 155–166Google Scholar
  33. Morris RO, Bilyeu KD, Laskey JG, Cheikh NN. 1999. Isolation of a gene encoding a glycosylated cytokinin oxidase from maize. Biochem Biophys Res Commun 255:328–333CrossRefPubMedGoogle Scholar
  34. Motyka V, Faiss M, Strnad M, Kamínek M, Schmülling T. 1996. Changes in cytokinin content and cytokinin oxidase activity in response to derepression of ipt gene transcription in transgenic tobacco calli and plants. Plant Physiol 112:1035–1043PubMedGoogle Scholar
  35. Motyka V, Kamínek M. 1994. Cytokinin oxidase from auxin- and cytokinin-dependent callus cultures of tobacco (Nicotiana tabacum L.). J Plant Growth Regul 13:1–9CrossRefGoogle Scholar
  36. Motyka V, Vaňková R, Čapková V, Petrášek J, Kamínek M, and others. 2003. Cytokinin-induced upregulation of cytokinin oxidase activity in tobacco includes changes in enzyme glycosylation and secretion. Physiol Plant 117:11–21CrossRefGoogle Scholar
  37. Novák O, Tarkowski P, Tarkowská D, Doležal K, Lenobel R, and others. 2003. Quantitative analysis of cytokinins in plants by liquid chromatography—single-quadrupole mass spectrometry. Anal Chim Acta 480:207–218Google Scholar
  38. Pačes V, Kamínek M. 1976. Effect of ribosylzeatin isomers on the enzymatic degradation of N6-(Δ2-isopentenyl)adenosine. Nucleic Acids Res 3:2309–2314PubMedGoogle Scholar
  39. Palni LMS, Palmer MV, Letham DS. 1984. The stability and biological activity of cytokinin metabolites in soybean callus tissue. Planta 160:242–249CrossRefGoogle Scholar
  40. Parker CW, Letham DS, Gollnow BI, Summons RE, Duke CC, others. 1978. Regulators of cell division in plant tissues. XXV. Metabolism of zeatin by lupin seedlings. Planta 142:239–251CrossRefGoogle Scholar
  41. Sakakibara H. 2004. “Cytokinin biosynthesis and metabolism” In: Davies PJ (ed.), Plant Hormones. Biosynthesis, Signal Transduction, Action! Dordrecht, The Netherlands, Kluwer Academic Publishers, pp 95–114Google Scholar
  42. Schmülling T. 2002. New insights into the functions of cytokinins in plant development. J Plant Growth Regul 21:40–49PubMedGoogle Scholar
  43. Schmülling T, Werner T, Riefler M, Krupková E, Manns IBY. 2003. Structure and function of cytokinin oxidase/dehydrogenase genes of maize, rice, Arabidopsis and other species. J Plant Res 116:241–252PubMedGoogle Scholar
  44. Singh S, Letham DS, Jameson PE, Zhang R, Parker CW, and others. 1988. Cytokinin biochemistry in relation to leaf senescence. IV. Cytokinin metabolism in soybean explants. Plant Physiol 88:788–794CrossRefPubMedGoogle Scholar
  45. Singh S, Palni LMS, Letham DS. 1992. Cytokinin biochemistry in relation to leaf senescence. 5. Endogenous cytokinin levels and metabolism of zeatin riboside in leaf discs from green and senescent tobacco (Nicotiana rustica) leaves. J Plant Physiol 139:279–283Google Scholar
  46. Sondheimer E, Tzou D-S. 1971. The metabolism of hormones during seed germination and dormancy. II. The metabolism of 8-14C-zeatin in bean axes. Plant Physiol 47:516–520PubMedGoogle Scholar
  47. Strnad M. 1997. The aromatic cytokinins. Physiol Plant 101:674–688CrossRefGoogle Scholar
  48. Tarkowská D, Doležal K, Tarkowski P, Astot C, Holub J, and others. 2003. Identification of new aromatic cytokinins in Arabidopsis thaliana and Populus × canadensis leaves by LC-(+)ESI-MS and capillary liquid chromatography/frit-fast atom bombardment mass spectrometry. Physiol Plant 117:579–590PubMedGoogle Scholar
  49. Taylor NJ, Stirk WA, van Staden J. 2003. The elusive cytokinin biosynthetic pathway. South Afr J Bot 69:269–281Google Scholar
  50. Van Staden J, Forsyth C. 1986. The metabolism of adenine and zeatin in immature caryopses of maize. J Plant Physiol 124:299–308Google Scholar
  51. Vaseva-Gemisheva I, Lee D, Alexieva V, Karanov E. 2004. Cytokinin oxidase/dehydrogenase in Pisum sativum plants during vegetative development. Influence of UV-B irradiation and high temperature on enzymatic activity. Plant Growth Regul 42:1–5CrossRefGoogle Scholar
  52. Veach YK, Martin RC, Mok DWS, Malbeck J, Vankova R, and others. 2003. O-Glucosylation of cis-zeatin in maize. Characterization of genes, enzymes, and endogenous cytokinins. Plant Physiol 131:1374–1380CrossRefPubMedGoogle Scholar
  53. Werner T, Motyka V, Strnad M, Schmülling T. 2001. Regulation of plant growth by cytokinin. Proc Natl Acad Sci USA 98:10487–10492CrossRefPubMedGoogle Scholar
  54. Werner T, Motyka V, Laucou V, Smets R, Van Onckelen H, and others. 2003. Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. Plant Cell 15:2532–2550CrossRefPubMedGoogle Scholar
  55. Yang SH, Yu H, Goh CJ. 2002a. Functional characterisation of a cytokinin oxidase gene DSCKX1 in Dendrobium orchid. Plant Mol Biol 51:237–248Google Scholar
  56. Yang SH, Yu H, Goh CJ. 2002b. Isolation and characterization of the orchid cytokinin oxidase DSCKX1 promoter. J Exp Bot 53:1899–1907CrossRefGoogle Scholar
  57. Yonekura-Sakakibara K, Kojima M, Yamaya T, Sakakibara H. 2004. Molecular characterization of cytokinin-responsive histidine kinases in maize. Differential ligand preferences and response to cis-zeatin. Plant Physiol 134:1654–1661CrossRefPubMedGoogle Scholar
  58. Zažímalová E, Kamínek M, Březinová A, Motyka V. 1999. “Control of cytokinin biosynthesis and metabolism” In: Hooykaas PJJ, Hall MA, Libbenga KR (eds.) Biochemistry and Molecular Biology of Plant Hormones. Amsterdam, The Netherlands, Elsevier Science B.V., pp 141–160Google Scholar
  59. Zhang R, Letham DS. 1990. Cytokinin translocation and metabolism in lupin species. III. Translocation of xylem cytokinin into the seeds of lateral shoots of Lupinus angustifolius. Plant Sci 70:65–71CrossRefGoogle Scholar
  60. Zhang R, Letham DS, Willcocks DA. 2002. Movement to bark and metabolism of xylem cytokinins in stems of Lupinus angustifolius. Phytochemistry 60:483–488PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  • Alena Gaudinová
    • 1
  • Petre I. Dobrev
    • 1
  • Blanka Šolcová
    • 1
  • Ondřej Novák
    • 2
  • Miroslav Strnad
    • 2
  • David Friedecký
    • 3
  • Václav Motyka
    • 1
  1. 1.Institute of Experimental BotanyAcademy of Sciences of the Czech RepublicPrague 6Czech Republic
  2. 2.Laboratory of Growth Regulators, Palacký University & Institute of Experimental BotanyAcademy of Sciences of the Czech RepublicŠlechtitelůCzech Republic
  3. 3.Laboratory for Inherited Metabolic Disorders, Department of Clinical BiochemistryUniversity HospitalCzech Republic

Personalised recommendations